The activation of the sirtuin1 (SIRT1)/nuclear factor erythroid 2-related factor 2 (Nrf2)/heme oxygenase 1 (HO-1) pathway has been shown to mitigate oxidative stress-induced apoptosis and mitochondrial damage by reducing reactive oxygen species (ROS) levels. Clinical trials have demonstrated that Zhongfeng Xingnao Liquid (ZFXN) ameliorates post-stroke cognitive impairment (PSCI). However, the underlying mechanism, particularly whether it involves protecting mitochondria and inhibiting apoptosis through the SIRT1/Nrf2/HO-1 pathway, remains unclear. This study employed an oxygen-glucose deprivation (OGD) cell model using SH-SY5Y cells and induced PSCI in rats through modified bilateral carotid artery ligation (2VO). The effects of ZFXN on learning and memory, neuroprotective activity, mitochondrial function, oxidative stress, and the SIRT1/Nrf2/HO-1 pathway were evaluated both in vivo and in vitro. Results indicated that ZFXN significantly increased the B-cell lymphoma 2 (Bcl2)/Bcl2-associated X (Bax) ratio, reduced terminal deoxynucleotidyl transferase-mediated dUTP nick-end-labeling (TUNEL)⁺ cells, and markedly improved cognition, synaptic plasticity, and neuronal function in the hippocampus and cortex. Furthermore, ZFXN exhibited potent antioxidant activity, evidenced by decreased ROS and malondialdehyde (MDA) content and increased superoxide dismutase (SOD), catalase (CAT), and glutathione (GSH) levels. ZFXN also demonstrated considerable enhancement of mitochondrial membrane potential (MMP), Tom20 fluorescence intensity, adenosine triphosphate (ATP) and energy charge (EC) levels, and mitochondrial complex I and III activity, thereby inhibiting mitochondrial damage. Additionally, ZFXN significantly increased SIRT1 activity and elevated SIRT1, nuclear Nrf2, and HO-1 levels. Notably, these effects were substantially counteracted when SIRT1 was suppressed by the inhibitor EX-527 in vitro. In conclusion, ZFXN alleviates PSCI by activating the SIRT1/Nrf2/HO-1 pathway and preventing mitochondrial damage.
Sirtuin 1/genetics* ; Animals ; NF-E2-Related Factor 2/genetics* ; Cognitive Dysfunction/genetics* ; Male ; Rats, Sprague-Dawley ; Rats ; Humans ; Signal Transduction/drug effects* ; Drugs, Chinese Herbal/administration & dosage* ; Heme Oxygenase-1/genetics* ; Stroke/complications* ; Oxidative Stress/drug effects* ; Apoptosis/drug effects* ; Mitochondria/metabolism* ; Reactive Oxygen Species/metabolism* ; Neuroprotective Agents

Ischemic stroke (IS) is a prevalent neurological disorder often resulting in significant disability or mortality. Resveratrol, extracted from Polygonum cuspidatum Sieb. et Zucc. (commonly known as Japanese knotweed), has been recognized for its potent neuroprotective properties. However, the neuroprotective efficacy of its derivative, (E)-4-(3,5-dimethoxystyryl) quinoline (RV02), against ischemic stroke remains inadequately explored. This study aimed to evaluate the protective effects of RV02 on neuronal ischemia-reperfusion injury both in vitro and in vivo. The research utilized an animal model of middle cerebral artery occlusion/reperfusion and SH-SY5Y cells subjected to oxygen-glucose deprivation and reperfusion to simulate ischemic conditions. The findings demonstrate that RV02 attenuates neuronal mitochondrial damage and scavenges reactive oxygen species (ROS) through mitophagy activation. Furthermore, Parkin knockdown was found to abolish RV02's ability to activate mitophagy and neuroprotection in vitro. These results suggest that RV02 shows promise as a neuroprotective agent, with the activation of Parkin-mediated mitophagy potentially serving as the primary mechanism underlying its neuroprotective effects.
Animals ; Ubiquitin-Protein Ligases/genetics* ; Mitophagy/drug effects* ; Resveratrol/analogs & derivatives* ; Neuroprotective Agents/pharmacology* ; Humans ; Neurons/metabolism* ; Reactive Oxygen Species/metabolism* ; Ischemic Stroke/genetics* ; Male ; Quinolines/pharmacology* ; Mice ; Fallopia japonica/chemistry* ; Mitochondria/metabolism* ; Reperfusion Injury/metabolism* ; Rats ; Mice, Inbred C57BL ; Disease Models, Animal

Triple-negative breast cancer (TNBC) represents an aggressive breast cancer subtype with poor prognosis and limited targeted treatment options. This investigation examined the anti-cancer potential of Caerulomycin A (Cae A), a natural compound derived from marine actinomycetes, against TNBC. Cae A demonstrated selective inhibition of viability and proliferation in TNBC cell lines, including 4T1, MDA-MB-231, and MDA-MB-468, through apoptosis induction. Mechanistic analyses revealed that the compound induced sustained endoplasmic reticulum (ER) stress and subsequent upregulation of C/EBP homologous protein (CHOP) expression, resulting in mitochondrial damage-mediated apoptosis. Inhibition of ER stress or CHOP expression knockdown reversed mitochondrial damage and apoptosis, highlighting the essential role of ER stress and CHOP in Cae A's anti-tumor mechanism. Both oxygen consumption rate (OCR) and extracellular acidification rate (ECAR) decreased in TNBC cells following Cae A treatment, indicating reduced mitochondrial respiratory and glycolytic capacities. This diminished energy metabolism potentially triggers ER stress and subsequent apoptosis. Furthermore, Cae A exhibited significant anti-tumor effects in the 4T1 tumor model in vivo without apparent toxicity. The compound also effectively inhibited human TNBC organoid growth. These results indicate that Cae A may serve as a potential therapeutic agent for TNBC, with its efficacy likely mediated through the disruption of glucose metabolism and the induction of ER stress-associated apoptosis.
Humans ; Endoplasmic Reticulum Stress/drug effects* ; Triple Negative Breast Neoplasms/genetics* ; Apoptosis/drug effects* ; Cell Line, Tumor ; Female ; Animals ; Glucose/metabolism* ; Mice ; Cell Proliferation/drug effects* ; Transcription Factor CHOP/genetics* ; Antineoplastic Agents/pharmacology* ; Mitochondria/metabolism* ; Mice, Inbred BALB C

Triclocarban (TCC) is a broad-spectrum antimicrobial widely used in various personal care products, textiles, and children's toys. TCC has potential reproductive and developmental toxicity in animals. However, little is known regarding the effect of TCC on human sperm function. In this study, an in vitro assay was used to investigate the effects of TCC on normal human spermatozoa and the possible underlying mechanisms involved. Semen from healthy male donors was collected and cultured in complete Biggers, Whitten and Whittingham (BWW) and low-sugar BWW media, followed by treatment with TCC at concentrations of 0, 0.1 µmol l -1 , 1 µmol l -1 , 10 µmol l -1 , and 100 µmol l -1 for 4 h. TCC was found to reduce the sperm total motility and progressive motility. Moreover, the sperm kinematic parameters, straight-line velocity (VSL), average path velocity (VAP), and curvilinear velocity (VCL) were affected in a dose-dependent manner. After treatment with TCC at the lowest effective concentration of 10 µmol l -1 , TCC caused a significant decrease in mitochondrial adenosine triphosphate (ATP) production and mitochondrial membrane potential (MMP) and a significant increase in reactive oxygen species (ROS), similar to the observations with the positive control carbonyl cyanide 4-(trifluoromethoxy) phenylhydrazone (FCCP), suggesting that TCC may decrease sperm motility by affecting the oxidative phosphorylation (OXPHOS) pathway. In a sugar-free and low-sugar BWW culture environment, TCC enhanced the damaging effect on sperm motility and ATP, MMP, and lactate decreased significantly, suggesting that TCC may also affect the glycolytic pathway that supplies energy to spermatozoa. This study demonstrates a possible mechanism of TCC toxicity in spermatozoa involving both the OXPHOS and glycolysis pathways.
Male ; Sperm Motility/drug effects* ; Humans ; Carbanilides/pharmacology* ; Oxidative Phosphorylation/drug effects* ; Glycolysis/drug effects* ; Membrane Potential, Mitochondrial/drug effects* ; Adenosine Triphosphate/metabolism* ; Spermatozoa/metabolism* ; Reactive Oxygen Species/metabolism* ; Mitochondria/metabolism*

OBJECTIVE: To investigate the inhibitory effect of Tanreqing Injection (TRQ) on the activation of nucleotide-binding oligomerization domain-like receptor pyrin domain containing 3 (NLRP3) inflammasome in macrophages infected with influenza A virus and the underlying mechanism based on mitophagy pathway. METHODS: The inflammatory model of murine macrophage J774A.1 induced by influenza A virus [strain A/Puerto Rico/8/1934 (H1N1), PR8] was constructed and treated by TRQ, while the mitochondria-targeted antioxidant Mito-TEMPO and autophagy specific inhibitor 3-methyladenine (3-MA) were used as controls to intensively study the anti-inflammatory mechanism of TRQ based on mitophagy-mitochondrial reactive oxygen species (mtROS)-NLRP3 inflammasome pathway. The levels of NLRP3, Caspase-1 p20, microtubule-associated protein 1 light chain 3 II (LC3II) and P62 proteins were measured by Western blot. The release of interleukin-1β (IL-1β) was tested by enzyme linked immunosorbent assay, the mtROS level was detected by flow cytometry, and the immunofluorescence and co-localization of LC3 and mitochondria were observed under confocal laser scanning microscopy. RESULTS: Similar to the effect of Mito-TEMPO and contrary to the results of 3-MA treatment, TRQ could significantly reduce the expressions of NLRP3, Caspase-1 p20, and autophagy adaptor P62, promote the expression of autophagy marker LC3II, enhance the mitochondrial fluorescence intensity, and inhibit the release of mtROS and IL-1β (all P<0.01). Moreover, LC3 was co-localized with mitochondria, confirming the type of mitophagy. CONCLUSION TRQ could reduce the level of mtROS by promoting mitophagy in macrophages infected with influenza A virus, thus inhibiting the activation of NLRP3 inflammasome and the release of IL-1β, and attenuating the inflammatory response.
Mitophagy/drug effects* ; NLR Family, Pyrin Domain-Containing 3 Protein/metabolism* ; Animals ; Macrophages/virology* ; Inflammasomes/drug effects* ; Drugs, Chinese Herbal/pharmacology* ; Mice ; Mitochondria/metabolism* ; Reactive Oxygen Species/metabolism* ; Influenza A virus/physiology* ; Interleukin-1beta/metabolism* ; Cell Line ; Injections

OBJECTIVE: To investigate the therapeutic potential of pseudolaric acid B (PAB) on non-alcoholic fatty liver disease (NAFLD) and its underlying molecular mechanism in vitro and in vivo. METHODS: Eight-week-old male C57BL/6J mice (n=32) were fed either a normal chow diet (NCD) or a high-fat diet (HFD) for 8 weeks. The HFD mice were divided into 3 groups according to a simple random method, including HFD, PAB low-dose [10 mg/(kg·d), PAB-L], and PAB high-dose [20 mg/(kg·d), PAB-H] groups. After 8 weeks of treatment, glucose metabolism and insulin resistance were assessed by oral glucose tolerance test (OGTT) and insulin tolerance test (ITT). Biochemical assays were used to measure the serum and cellular levels of total cholesterol (TC), triglycerides (TG), aspartate aminotransferase (AST), alanine aminotransferase (ALT), low-density lipoprotein cholesterol (LDL-C), and high-density lipoprotein cholesterol (HDL-C). White adipose tissue (WAT), brown adipose tissue (BAT) and liver tissue were subjected to hematoxylin and eosin (H&E) staining or Oil Red O staining to observe the alterations in adipose tissue and liver injury. PharmMapper and DisGeNet were used to predict the NAFLD-related PAB targets. Peroxisome proliferator-activated receptor alpha (PPARα) pathway involvement was suggested by Kyoto Encyclopedia of Genes and Genomes (KEGG) and search tool Retrieval of Interacting Genes (STRING) analyses. Luciferase reporter assay, cellular thermal shift assay (CETSA), and drug affinity responsive target stability assay (DARTS) were conducted to confirm direct binding of PAB with PPARα. Molecular dynamics simulations were applied to further validate target engagement. RT-qPCR and Western blot were performed to assess the downstream genes and proteins expression, and validated by PPARα inhibitor MK886. RESULTS: PAB significantly reduced serum TC, TG, LDL-C, AST, and ALT levels, and increased HDL-C level in HFD mice (P<0.01). Target prediction analysis indicated a significant correlation between PAB and PPARα pathway. PAB direct target binding with PPARα was confirmed through luciferase reporter assay, CETSA, and DARTS (P<0.05 or P<0.01). The target engagement between PAB and PPARα protein was further confirmed by molecular dynamics simulations and the top 3 amino acid residues, LEU321, MET355, and PHE273 showed the most significant changes in mutational energy. Subsequently, PAB upregulated the genes expressions involved in lipid metabolism and mitochondrial biogenesis downstream of PPARα (P<0.05 or P<0.01). Significantly, the PPARα inhibitor MK886 effectively reversed the lipid-lowering and PPARα activation properties of PAB (P<0.05 or P<0.01). CONCLUSION PAB mitigates lipid accumulation, ameliorates liver damage, and improves mitochondrial biogenesis by binding with PPARα, thus presenting a potential candidate for pharmaceutical development in the treatment of NAFLD.
Animals ; PPAR alpha/metabolism* ; Non-alcoholic Fatty Liver Disease/pathology* ; Male ; Mice, Inbred C57BL ; Lipid Metabolism/drug effects* ; Diterpenes/therapeutic use* ; Organelle Biogenesis ; Diet, High-Fat ; Humans ; Mice ; Liver/metabolism* ; Insulin Resistance ; Mitochondria/metabolism* ; Molecular Docking Simulation

OBJECTIVES: To investigate the protective effect of Yiqi Yangyin Huazhuo Tongluo Formula (YYHT) against high glucose-induced injury in mouse renal podocytes (MPC5 cells) and the possible mechanism. METHODS: Adult Wistar rats were treated with 19, 38, and 76 g/kg YYHT or saline via gavage for 7 days to prepare YYHT-medicated or blank sera for treatment of MPC5 cells cultured in high glucose (30 mmol/L) prior to transfection with a miR-21a-5p inhibitor or a miR-21a-5p mimic. The changes in miR-21a-5p expressions and the mRNA levels of FoxO1, PINK1, and Parkin in the treated cells were detected with qRT-PCR, and the protein levels of nephrin, podocin, FoxO1, PINK1, and Parkin were detected with Western blotting. Autophagic activity in the cells were evaluated with MDC staining. The effect of miR-21a-5p mimic on FoxO1 transcription and the binding of miR-21a-5p to FoxO1 were examined with luciferase reporter gene assay and radioimmunoprecipitation assay. RESULTS: MPC5 cells exposed to high glucose showed significantly increased miR-21a-5p expression, lowered expressions of FoxO1, PINK1, and Parkin1 mRNAs, and reduced levels of FoxO1, PINK1, parkin, nephrin, and podocin proteins and autophagic activity. Treatment of the exposed cells with YYHT-medicated sera and miR-21a-5p inhibitor both significantly enhanced the protein expressions of nephrin and podocin, inhibited the expression of miR-21a-5p, increased the mRNA and protein expressions of FoxO1, PINK1 and Parkin, and upregulated autophagic activity of the cells. Transfection with miR-21a-5p mimic effectively inhibited the transcription of FoxO1 and promoted the binding of miR-21a-5p to FoxO1 in MPC5 cells, and these effects were obviously attenuated by treatment with YYHT-medicated sera. CONCLUSIONS YYHT-medicated sera alleviate high glucose-induced injury in MPC5 cells by regulating miR-21a-5p/FoxO1/PINK1-mediated mitochondrial autophagy.
Animals ; MicroRNAs/genetics* ; Podocytes/pathology* ; Drugs, Chinese Herbal/pharmacology* ; Autophagy/drug effects* ; Rats, Wistar ; Protein Kinases/metabolism* ; Rats ; Forkhead Box Protein O1 ; Mice ; Mitochondria/drug effects* ; Ubiquitin-Protein Ligases/metabolism* ; Glucose ; Diabetic Nephropathies ; Male ; Membrane Proteins/metabolism* ; Intracellular Signaling Peptides and Proteins

OBJECTIVES: To investigate the role of the BNIP3-PI3K/Akt signaling pathway in mediating the inhibitory effect of Buyang Huanwu Decoction (BYHWT) on mitochondrial autophagy in human synovial fibroblasts from rheumatoid arthritis patients (FLS-RA) cultured under a hypoxic condition. METHODS: Forty normal Wistar rats were randomized into two groups (n=20) for daily gavage of BYHWT or distilled water for 7 days to prepare BYHWT-medicated or control sera. FLS-RA were cultured in routine condition or exposed to hypoxia (10% O₂) for 24 h wigh subsequent treatment with IL-1β, followed by treatment with diluted BYHWT-medicated serum (5%, 10% and 20%) or control serum. AnnexinV-APC/7-AAD double staining and T-AOC kit were used for detecting apoptosis and total antioxidant capacity of the cells, and the changes in ROS, ATP level, mitochondrial membrane potential and Ca²⁺ homeostasis were analyzed. The changes in mRNA and protein expressions of BNIP3, PI3K and AKT and mRNA expressions of LC3, Beclin-1 and P62 were detected using RT-qPCR and Western blotting. RESULTS: Treatment with BYHWT-medicated serum dose-dependently lowered apoptosis rate of IL-1β-induced FLS-RA with hypoxic exposure. The treatment significantly decreased T-AOC concentration, increased ROS production, autophagosome formation and ATPase levels, and lowered mitochondrial membrane potential and Ca²⁺ level in the cells. In IL-1β-induced FLS-RA with hypoxic exposure, treatment with BYHWT-medicated serum significantly increased BNIP3 protein expression, decreased the protein expressions of PI3K and AKT, increased the mRNA expressions of BNIP3 and P62, and lowered the mRNA expressions of PI3K, AKT, LC3 and Beclin-1 without significantly affecting Beclin-1 protein expression. The cells treated with 5% and 10% BYHWT-medicated serum showed no significant changes in LC3 expression. CONCLUSIONS BYHWT inhibits mitochondrial autophagy in IL-1β-induced FLS-RA with hypoxic exposure possibly by inhibiting BNIP3-mediated PI3K/AKT signaling pathway.
Drugs, Chinese Herbal/pharmacology* ; Arthritis, Rheumatoid/pathology* ; Animals ; Signal Transduction/drug effects* ; Proto-Oncogene Proteins c-akt/metabolism* ; Autophagy/drug effects* ; Humans ; Fibroblasts/cytology* ; Rats, Wistar ; Membrane Proteins/metabolism* ; Rats ; Phosphatidylinositol 3-Kinases/metabolism* ; Mitochondria/metabolism* ; Cells, Cultured ; Proto-Oncogene Proteins/metabolism* ; Apoptosis/drug effects* ; Cell Hypoxia ; Synovial Membrane/cytology* ; Male ; Mitochondrial Proteins

OBJECTIVES: To explore the mechanism of Shenqi Buzhong (SQBZ) Formula for alleviating mitochondrial dysfunction in a rat model of chronic obstructive pulmonary disease (COPD) in light of the AMPK/SIRT1/PGC-1α pathway. METHODS: Fifty male SD rat models of COPD, established by intratracheal lipopolysaccharide (LPS) instillation, exposure to cigarette smoke, and gavage of Senna leaf infusion, were randomized into 5 groups (n=10) for treatment with saline (model group), SQBZ Formula at low, moderate and high doses (3.08, 6.16 and 12.32 g/kg, respectively), or aminophylline (0.024 g/kg) by gavage for 4 weeks, with another 10 untreated rats as the control group. Pulmonary function of the rats were tested, and pathologies and ultrastructural changes of the lung tissues were examined using HE staining and transmission electron microscopy. The levels of SOD, ATP, MDA, and mitochondrial membrane potential in the lungs were detected using WST-1, colorimetric assay, TBA, and JC-1 methods. Flow cytometry was used to analyze ROS level in the lung tissues, and the protein expression levels of P-AMPKα, AMPKα, SIRTI, and PGC-1α were detected using Western blotting. RESULTS: The rat models of COPD showed significantly decreased lung function, severe histopathological injuries of the lungs, decreased pulmonary levels of SOD activity, ATP and mitochondrial membrane potential, increased levels of MDA and ROS, and decreased pulmonary expressions of P-AMPKα, SIRTI, and PGC-1α proteins. All these changes were significantly alleviated by treatment with SQBZ Formula and aminophylline, and the efficacy was comparable between high-dose SQBZ Formula group and aminophylline group. CONCLUSIONS SQBZ Formula ameliorates mitochondrial dysfunction in COPD rats possibly by activating the AMPK/SIRT1/PGC-1α pathway.
Animals ; Pulmonary Disease, Chronic Obstructive/drug therapy* ; Drugs, Chinese Herbal/therapeutic use* ; Sirtuin 1/metabolism* ; Peroxisome Proliferator-Activated Receptor Gamma Coactivator 1-alpha ; Rats, Sprague-Dawley ; Male ; Rats ; AMP-Activated Protein Kinases/metabolism* ; Mitochondria/metabolism* ; Disease Models, Animal ; Signal Transduction/drug effects*

OBJECTIVES: To investigate the molecular mechanism by which salvianolic acid B (Sal-B) modulates mitochondrial functional homeostasis and alleviates myocardial ischemia-reperfusion (I/R) injury in mice. METHODS: Mouse cardiomyocyte HL-1 cells were pretreated with 5 μmol/L Sal-B with or without sh-Sirt1 transfection before exposure to hypoxia-reoxygenation (HR), and the changes in ATP production, mitochondrial superoxide activity, substrate oxidation level were evaluated. In the animal experiment, 36 C57BL/6J mice were randomized into 3 groups (n=12) for sham operation or ligation of the left anterior coronary artery to induce myocardial I/R injury with or without intravenous injection of Sal-B+I/R (50 mg/kg). In the rescue experiment, 60 adult C57BL/6J mice were randomized into 5 groups (n=12): sham-operated group, myocardial I/R group, Sal-B+I/R group, I/R+Sal-B+Sirt1fl/fl group, and I/R+Sal-B+cKO-Sirt1 group. Myocardial injury was evaluated with HE staining, and cardiac function was assessed by measurement of the ejection fraction and fractional shortening using echocardiography. RESULTS: In HL-1 cells with HR injury, Sal-B pretreatment significantly increased cellular ATP production, reduced mitochondrial superoxide anion levels, and enhanced oxygen consumption level. In the mouse models of myocardial I/R injury, Sal-B pretreatment markedly ameliorated I/R-induced structural disarray of the cardiac myocytes and improved cardiac ejection. Cycloheximide chase with Western blotting and ubiquitination assays after Sirt1-IP showed that Sal-B significantly inhibited Sirt1 degradation in HL-1 cells. Sirt1 knock-down reversed Sal-B-induced increases in ATP production, reduction in superoxide, and elevation of OCR in HL-1 cells. Cardiomyocyte-specific Sirt1 knockout obviously reversed Sal-B-mediated improvement in cardiac ejection function and myocardial structure damage in mice with myocardial I/R injury. CONCLUSIONS Sal-B promotes mitochondrial functional homeostasis in cardiomyocytes with HR injury and improves cardiac function in mice after myocardial I/R by inhibiting Sirt1 protein degradation.
Animals ; Sirtuin 1/metabolism* ; Myocardial Reperfusion Injury/physiopathology* ; Mice, Inbred C57BL ; Mice ; Myocytes, Cardiac/drug effects* ; Benzofurans/pharmacology* ; Homeostasis/drug effects* ; Male ; Mitochondria/drug effects* ; Depsides